Neurobiology of Aging, Vol. 10, pp. 463-465. © Pergamon Press plc. 1989. Printed in the U.S.A. 0197-4580/89 $3.00 + .00

COMMENTARIES

Potential Roles of Protease Inhibitors in Alzheimer's Disease

C A R M E L A R. A B R A H A M 1

Department of Neurobiology, Harvard Medical School, Boston, MA 02115

Recently, protease inhibitors have been recognized as potential contributors to the pathogenesis of Alzheimer's disease. In this role, they could mediate an exaggerated regenerative response in the brain, participate as acute phase reactants, or be involved in the aberrant proteolytic processing of the amyloid proteins. Protease inhibitors are, therefore, attractive targets for drug intervention in Alzheimer's disease.

ALTHOUGH we have been faced in the past few years with an explosion of new information about Alzheimer's disease genes and proteins, we are still far from being able to answer the major questions concerning the disease: 1) What are the primary gene defects in the familial cases and how do they affect neuronal cell loss and amyloid accumulation, 2) Why does the amyloid occur only late in life, and 3) Are the intracellular and extracellular amyloid deposits detrimental to the surrounding tissue? While answers to the first two questions are not yet in hand, they are being extensively investigated. Present information does, how- ever, allow us to begin to speculate about the solution to this last question.

In this regard, Caputo and Salama have written a comprehen- sive review on the amyloid proteins as potential targets for drug therapy, relying on the hypothesis that the mere physical existence of neurofibrillary tangles and amyloid in plaques and blood vessel walls contributes to cellular dysfunction, neuronal disconnection and ultimately cell death and cognitive deficits. The authors suggest two plausible explanations for the accumulation of the abnormal proteins in Alzheimer's disease: an overexpression of their genes or abnormal posttranslational processing events which render them into a new insoluble conformation. Either of these two pathologic processes, as discussed by the authors, would provide points for intervention in the treatment of Alzheimer's disease.

DO PROTEASE INHIBITORS MEDIATE AN EXAGGERATED REGENERATIVE RESPONSE?

I would like to take this opportunity to consider additional potential targets for drug therapy in Alzheimer's disease (AD). Starting in utero a percentage of our neurons undergo a pro- grammed cell death which continues throughout life. Normally when a neuron dies, it is engulfed by nearby cells and disinte- grates. Let us assume that in Alzheimer's disease there is an abnormal response to cell death in the form of compensatory cells that overproduce growth factors or neurite extension factors. Possible candidates for such factors include the [3-protein precur- sor (or parts of it) (27), as yet unknown factors found to be elevated in AD (26), serine proteases such as activated comple- ment factors found in plaques (5), serine protease inhibitors like ~l-antichymotrypsin (ACT) (1), a serine protease inhibitory do- main found in some transcripts of the [3-amyloid protein precursor

([3-APP) (11, 18, 25) or glycosaminoglycans (GAGS) (23). If the fine balance between all these molecules is lost, abnormal degra- dation patterns of the molecules involved may result, possibly leading to changes in conformation, including exposure of new epitopes, such that the proteins are now able to interact with surrounding molecules and cause extracellular deposition. Such a scenario may explain why we detect in the amyloid plaques [3-protein, ACT, GAGS and complement components. Also, the enhanced growth factor activity would explain the neuritic degen- eration/regeneration, the massive somatodendritic sprouting or "curly fibers" (8,12) and the extensive basilar dendrites of pyramidal neurons not seen in age-matched controls (13). Inevi- tably, this attempt to regenerate would cause exhaustion and death of the neurons involved. If the above picture describes what actually occurs in AD, we can design drugs aimed at reducing this regeneration. Of course, we would not be attacking the primary cause of the cell death, but such treatment may alleviate some of the symptoms and arrest the disease.

DOES THE ACUTE PHASE RESPONSE PLAY A ROLE IN ALZHEIMER'S DISEASE?

Proteases and protease inhibitors may not only play an impor- tant role in neurite extension as discussed elsewhere (1), but also take part in the acute phase response. For example, we found the expression of ACT to be elevated in AD gray matter, as compared to control. Astrocytes appear to be the major producers of ACT in the brain as shown by in situ hybridization (J. Pasternack et al.; E. Koo, et al., unpublished results). We previously postulated that some of the pathology in AD may be a result of an acute phase response of the brain with the astrocytes being important players (2). Since ACT is an acute phase protein and the promoter of the [3-APP gene was also shown to contain a heat shock control element (22), it is conceivable that both are up-regulated in response to stress situations. In secondary amyloidosis, where fragments of the serum amyloid A protein (SAA) precipitates as amyloid, SAA is also up-regulated in response to a variety of inflammatory or toxic conditions (4). ACT expression in the liver can be up-regulated by IL-1 (3) and recently it has been shown by immunocytochemistry that astrocytes in AD and Down's syn- drome (DS) overexpress IL-1 (S. Griffin, personal communica- tion). Also, [3-APP expression can be induced with IL-1 (D.

1Requests for reprints should be addressed to Dr. Carmela Abraham. Arthritis Center K-5, Boston University School of Medicine, 71 East Concord Street, Boston, MA 02118.

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Goldgaber, personal communication). Thus, it seems that drugs that could specifically inhibit the inflammatory response in the brain, by reducing the amounts of IL-1 or other, yet unknown, modulators could also prove helpful in arresting the symptoms in Alzheimer's disease.

PROTEOLYTIC DEGRADATION OF INTRACELLULAR AND EXTRACELLULAR AMYLOID

Two independent findings suggest that Alzheimer's amyloid deposits accumulate as a result of aberrant degradation. Intracel- lularly, the paired helical filaments found in neuronal perikarya and in the neurites of neuritic plaques, contain ubiquitin (15,16). Ubiquitin is a 8 Kd protein which identifies abnormal cellular proteins and covalently binds to them, marking them for ATP- dependent proteolysis. In AD, the ubiquitin-conjugate degrading system may be faulty and the tangles persist, even after the death of the neuron, as "ghost tangles."

Extracellularly, the [3-protein amyloid accumulates in the center of senile plaques and in intracortical and meningeal blood vessels. The 13-protein is a 4 Kd fragment (6) of a much larger protein precursor (7, 10, 21, 24), Under normal conditions the 13-protein either does not occur or is cleared at the same rate that it is formed. In AD, DS and normal aging of humans and other mammals the 13-protein accumulates, presumably as a result of abnormal degradation of the precursor. Recently, it has been shown that the vascular amyloid in AD is only 39 amino acids long

(20) similar to the amyloid of hereditary cerebral hemorrhage with amyloidosis of Dutch origin (19). C. Joachim and her colleagues also sequenced the meningovascular amyloid and found it to be 40 amino acids long (9). In contrast, the plaque core amyloid was shown to be comprised of 42-43 residues (14). This 3--4 amino acid difference may indicate differential proteolytic processing in the two milieus, may explain the different solubility properties of the two amyloids, and may determine how the tissue reacts to both forms. It is conceivable that only the 42-43 amino acid form induces a neuritic response, since in the Dutch disease, where the 39 amino acid form of 13-protein was described, only diffuse plaques form. In this regard it will be interesting to compare the length of the [3-protein in the diffuse plaques of the cerebellum and striatum in AD, where neuritic degeneration or dysfunction is not seen, with amyloid in cortical or limbic plaques. The next step will be to identify the tissue-specific proteases and protease inhibitors responsible for the [3-APP degradation. If an enzyme whose activity results in the formation of the 13-protein is identified, drugs that can cross the blood-brain barrier and inhibit it can be tried in macaque monkeys which were shown to have an identical [3-APP to humans (17).

In summary, proteases and protease inhibitors may play a role in the pathogenesis of Alzheimer's disease at several levels; as part of the neuritic extension, as part of an acute phase response in the brain, or directly on the formation of the intracellular and extracellular amyloid deposits. All three levels can be considered potential targets for drug therapy, even before the discovery of the primary genetic defects in Alzheimer's disease.

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2. Abraham, C. R.; Potter, H. Alzheimer's disease: recent advances in understanding the brain amyloid deposits. Biotechnology 7:147-153; 1989.

3. Baumann, H.; Richards, C.; Gauldie, J. Interaction among hepato- cyte-stimulating factors, interleukin 1, and glucocorticoids for regu- lation of acute phase plasma proteins in human hepatoma (HepG2) ceils. J. Immunol. 139:4122-4178; 1987.

4. Benditt, E. P.; Eriksen, N. Chemical classes of amyloid substance. Am. J. Pathol. 65:231-249; 1971.

5. Eikelenboom, P.; Hack, C. E.; Rozemuller, J. M.; Stam, F. C. Complement activation in amyloid plaques in Alzheimer's dementia. Virchows Arch. [B] 56:259-262; 1989.

6. Glenner, G. G.; Wong, C. W, Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Commun. 122:885-890; 1984.

7. Gotdgaber, D.; Lea-man, M. J.; McBride, O. W.; Saffiotti, V.; Gadjusek, D. C. Characterization and chromosomal localization of a cDNA encoding brain amyloid of Alzheimer's disease. Science 235:877; 1987.

8. Ihara, Y. Massive somatodendritic sprouting of cortical neurons in Alzheimer's disease. Brain Res. 459:138-144; 1988.

9. Joachim, C. L.; Duff)', L. K.; Morris, J. H.; Selkoe, D. J. Protein chemical and immunocytochemical studies of meningovascular t3- amyloid protein in Alzheimer's disease and normal aging. Brain Res. 474:100-111; 1988.

10. Kang, J.; Lemaire, H. G.; Unterback, A.; Salbaum, J. M.; Masters, C. L.; Grezeschik, K. H.; Multhaup, G.; Beyreuther, K.; Muller-Hill, B. The precursor of Alzheimer disease amyloid A4 protein resembles a cell-surface receptor. Nature 325:733; 1987.

11. Kitaguchi, N.; Takahashi, Y.; Tokushima, Y.; Shiojiri, S.; Ito, H. Novel precursor of Alzheimer's disease amyloid protein shows pro- tease inhibitory activity. Nature 331:530-532; 1988.

12. Kowall, N. W.; Kosik, K. S. Axonal disruption and aberrant localization of tau protein characterize the neuronal pathology of

Alzheimer's disease. Ann. Neurol. 22:639-643; 1987. 13. McKee, A. C.; Kowall, N. W.; Kosik, K. S. Micrntublar reorgani-

zation and growth response in Alzheimer's disease. Ann. Neurol.; in press.

14. Masters, C. L.; Simms, G.; Weinmann, N. A.; Multhaup, G.; McDonald, B. L.; Beyreuther, K. Amyloid plaque core protein in Alzheimer's disease and Down's syndrome. Proc. Natl. Acad, Sci. USA 82:4245-4249; 1985.

15. Mori, H.; Kondo, J.; Ihara, Y. Ubiquitin is a component of paired helical filaments in Alzheimer's disease. Science 235:1641-1644; 1987.

16. Perry, G.; Friedman, R.; Shaw, G.; Chau, V. Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites of Alzheimer's disease brains. Proc. Natl. Acad. Sci. USA 84:3033-3036; 1987.

17. Podlisny, M.; Gronbeck, A.; Tolan, D.; Oltersdorf, T.; Selkoe, D. Studies of the processing of 13-amytoid precursor in human, monkey and rodent brain and in cDNA-transfected cells. Alzheimer Dis. Assoc. Disord. 3(Suppl. 1):38; 1989.

18. Ponte, P.; Gonzalez-DeWhitt, P.; Schilling, J.; Miller, J.; Hsu, D.; Greenberg, B.; Davis, K.; Wallace, W.; Lieberburg, I.; Fuller, F.; Cordell, B. A new A4 amyloid mRNA contains a domain homologous to serine protease inhibitors. Nature 331:525-527; 1988.

19. Prelli, F.; Castano, E. M.; van Duinen, S. G.; Bots, G: Th. A. M.; Luyendijk, W.; Frangione, B. Different processing of Alzheimer's 13-protein precursor in the vessel wail of patients with hereditary. cerebral hemorrhage with amyloidosis--Dutch type. Biochem. Bin- phys. Res. Commun. 151:1150-1155; 1988.

20. Prelli, F.; Castano, E. M.; Gleaner, G. G.; Frangione, B. Differences between vascular and plaque core amyloid in Alzheimer's disease. J. Neurocbem. 51:648-651; 1988.

21. Robakis, N. K.; Ramakrishna, N.; Wolfe, G.; Wisniewski, H. M. Molecular cloning and characterization of a eDNA encoding the cerebrovascular and the neuritic plaque amyloid peptides. Proc. Natl. Acad. Sci. USA 84:4190--4194; 1987.

22. Salbaum, J. M.; Weidemann, A.; Lemaire, H.-G.; Masters, C. L.; Beyreuther, K. The promoter of Alzheimer's disease A4 precursor gene. EMBO J. 7:2807-2813; 1988.

23. Snow, A. D.; Willmer, J. P.; Kisilevsky, R. Sulfated glycosamino-

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glycans in Alzheimer's disease. Hum. Pathol. 18:506-510; 1987. 24. Tanzi, R. E.; Gusella, J. F.; Watkins, P. C.; Bruns, G. A. P.; St.

George-Hyslop, P.; Van Keuren, M. L.; Patterson, D.; Pajan, S.; Kumit, D. M.; Neve, R. L. Amyloid 13-protein gene; cDNA, mRNA distributions, and genetic linkage near the Alzheimer locus. Science 235:880-883; 1987.

25. Tanzi, R. E.; McClatchey, A. I.; Lamberti, E. D.; Villa-Komaroff, L.; Gusella, J. F.; Neve, R. L. Protease inhibitor domain encoded by an amyloid protein precursor mRNA associated with Alzheimer's

disease. Nature 331:528-530; 1988. 26. Uchida, Y,; Ihara, Y.; Tomonaga, M. Alzheimer's disease brain

extract stimulates the survival of cerebral cortical neurons from neonatal rats. Biochem. Biophys. Res. Commun. 150:1263-1267; 1988.

27. Whitson, J. S.; Selkoe, D. J.; Cotman, C. W. Amyloid 13 protein enhances the survival of hippocampal neurons in vitro. Science 243:1488-1490; 1989.

Zeroing in on Amyloid Proteins in Alzheimer Disease Therapy

B A R R Y W. F E S T O F F

Kansas City Veterans Administration Medical Center, Neurobiology 151 4801 Linwood Boulevard, Kansas City, MO 64128

Department of Neurology, University of Kansas Medical Center

The authors have provided us with a complete review of the approaches to the amyloid proteins of Alzheimer's disease in regards to targets for drug therapy. Sufficient information is now available concerning systemic amyloidogenesis, genes for familial Alzheimer's disease and the beta amyloid precursor protein, as well as the posttranslational processing requirements for amyloidogenesis and its prevention. Recent excitement concerning the roles for serine proteases and their inhibitors, both in the production and prevention of amyloidogenesis, make this review and its publication in the Neurobiology of Aging extremely timely.

CONSIDERABLE insight has been acquired quite recently in the biochemistry and molecular biology of the amyloid proteins that accumulate and concentrate in the brains of Alzheimer's disease (AD) victims. To be sure, we are still far from understanding the pathogenesis of brain amyloid formation, not to mention its relevance to the clinical symptomatology of AD, but enthusiasm for pursuing such approaches is growing in the scientific commu- nity. In their review, Drs. Caputo and Salama have provided us with intriguing possibilities of utilizing information, both positive and negative, gained from treatment approaches to the systemic amyloidoses, including familial amyloidotic polyneuropathy. This has been coupled with an eloquent and erudite discussion of the various approaches now extant in the emerging field of gene therapy, relevant to amyloid fibril formation.

A particularly intriguing, but controversial approach, briefly discussed by the authors, is the use of antisense DNA or antisense RNA in the modification of gene expression. Theoretically, such approaches have considerable merit. For instance, the utilization of DNA-binding proteins to regulate transcription of the APP gene takes on greater significance with the identification of the pro- moter region of the beta amyloid protein gene. It is of great interest that activator protein-1 (AP-l), acute phase proteins, nerve growth factor (NGF) and interleukin-1 (IL-1) are the promoter region DNA-binding proteins that may regulate transcription of the APP gene (see Caputo and Salama, this volume). In addition, research directed at blocking gene expression by complementary antisense RNA oligonucleotides binding to regulatory genes and, thereby, inhibiting specific RNA translation, may be an important avenue, although such research in amyloid formation has not, as yet, been reported. Similarly, antisense DNA, to bind to the gene segment directly in order to prevent transcription, although attractive, is still in the theoretical stage regarding amyloid formation.Clearly,

concerns regarding the extent to which the normal gene product might be manipulated take on special relevance with these ap- proaches.

One particularly promising area of therapeutic targeting in AD involves serine proteases and their natural inhibitors, known now as the serpins (3,5). These are important to AD research because of the existence of a sequence domain within the APP which is homologous to the Kunitz type of serpins [(4); see Caputo and Salama, this volume], The classic Kunitz inhibitor is aprotinin (Trasylol), a 6500 Mr polypeptide originally isolated from pan- creas, which is a very effective plasmin and kallikrein inhibitor, despite its original designation as the bovine pancreatic trypsin inhibitor (BPTI). The Kunitz domain, a 53 residue domain contained within the extracellular region of the APP, 350 or more residues towards the amino terminal from the transmembrane domain, is a potential focus for AD therapeutic approaches. Importantly, the general area of serine proteases and their inhibi- tors is extremely well-known. However, BPTI or aprotinin is different, in at least one respect, from the larger Mr serpins (50-100 kDa proteins), since once they bind their respective target protease, and a portion of their amino termini are cleaved, they complex the enzyme which cannot extricate itself and is, thus, inhibited. If the larger serpins should be released, they are significantly altered and usually devoid of further inhibitory activity. Aprotinin, as with other small inhibitors, is not cleaved and since it is already a small polypeptide, a molecule of it may be free to inhibit again and again, after forming a complex with a protease.

Another protease inhibitor of the serpin family has been implicated in AD. Using immunocytochemical techniques one laboratory has demonstrated the presence of alphat-antichymo- trypsin (cq-ACT) in amyloid plaques in AD patients' brains, as